Abstract/Summary

We have previously identified allosteric modulators of the cannabinoid CB1 receptor (Org 27569, PSNCBAM-1) which display a contradictory pharmacological profile:
increasing the specific binding of the CB1 receptor agonist [3H]CP55940 but producing a decrease in CB1 receptor agonist efficacy. Here we investigated the effect
one or both compounds in a broad range of signalling endpoints linked to CB1 receptor activation. We assessed the effect of these compounds on CB1 receptor
agonist-induced [35S]GTPγS binding, inhibition and stimulation of forskolin stimulated cAMP production, phosphorylation of ERK, and β arrestin recruitment.
We also investigated the effect of these allosteric modulators on CB1 agonist binding kinetics. Both compounds display ligand dependence, being significantly more potent
as modulators of CP55940 signalling as compared to WIN55212 and having little effect on [3H]WIN55212 binding. Org 27569 displays biased antagonism whereby it inhibits: agonist-induced [35S]GTPγS binding, simulation (Gαs mediated) and
inhibition (Gαi mediated) of cAMP production and β arrestin recruitment. In contrast, it acts as an enhancer of agonist-induced ERK phosphoryation. Alone, the compound can act also as an allosteric agonist, increasing cAMP production and ERK phosphorylation. We find that in both saturation and kinetic binding experiments, the Org 27569 and PSNCBAM-1 appeared to influence only orthosteric ligand maximum occupancy rather than affinity. The data indicate that the allosteric modulators share a common mechanism whereby they increase available high affinity CB1 agonist binding sites. The receptor conformation stabilised by the allosterics appears to induce signalling and also selectively traffics orthosteric agonist signalling via the ERK phosphorylation pathway.